Yersinia pestis causes human and animal plague. Plague vaccine research has increasing importance since Y. pestis is a potential bioterrorism agent. Published Y. pestis vaccine studies use mortality as the primary endpoint. Mortality endpoints have limited statistical limited power. Since mice are highly susceptible to plague endotoxin and exotoxin, murine mortality may be a poor predictor for human vaccine efficacy. Lastly, lethal challenge models lead to pain and suffering in experimental animals. We will evaluate alternative endpoints to be used in place of mortality in plague challenge models. The first two Aims include the adoption of standard subcutaneous and aerosol models of Y. pestis infection and daily measurement of multiple endpoints. Routine endpoints will include mortality, survival time, CFU in multiple sites, and histopathology. Novel endpoints (for plague models) will include comprehensive cytokine profiles and the characterization of host cells associated in vivo with Yersinia bacteria. We hope to learn more about plague pathophysiology, and identify better endpoints for measurement of vaccine efficacy. In the third Aim, we will validate selected endpoints by varying components of the challenge models: Y. pestis dose, Y. pestis challenge strain virulence, active or passive immunization, and nonspecific immune stimulation. Ideal endpoints will have one or more of the following characteristics: (1) consistent within groups, (2) large dynamic range, (3) identifiable early in the course of infection, (4) correlated with input variables (dose, virulence, active or passive immunization, nonspecific immune stimulation) and (5) correlated with mortality. We will evaluate specific hypotheses about the cytokine profiles (early interferon-gamma and tumor necrosis factor-alpha) and responding cell populations (early infiltration of neutrophils, macrophages, and CD69+ cells) associated with protection. We will use funding from this exploratory R21 application to complete the first two Aims and to begin the experiments in Aim 3. We will use the data generated to refine our hypotheses and to pursue further funding to complete Aim 3.